Polymers for separation of biomolecules by capillary electrophoresis

ABSTRACT

The invention provides uncharged water-soluble silica-adsorbing polymers for suppressing electroendoosmotic flow and to reduce analyte-wall interactions in capillary electrophoresis. In one aspect of the invention, one or more of such polymers are employed as components of a separation medium for the separation of biomolecules, such as polynucleotides, polysaccharides, proteins, and the like, by capillary electrophoresis. Generally, such polymers are characterized by (i) water solubility over the temperature range between about 20° C. to about 50° C., (ii) concentration in a separation medium in the range between about 0.001% to about 10% (weight/volume), (iii) molecular weight in the range of about 5×10 3  to about 1×10 6  daltons, and (iv) absence of charged groups in an aqueous medium having pH in the range of about 6 to about 9. In one embodiment, polymers of the invention are selected from the group consisting of polylactams, such as polyvinylpyrrolidone; N,N-disubstituted polyacrylamides; and N-substituted polyacrylamides. In accordance with the method of the invention, a sufficient amount of polymer adsorbs to the capillary surface to establish a zone of high viscosity that shields the analyte from the wall and impedes the movement of an electrical double layer under an electric field.

RELATED U.S. APPLICATIONS

This is a continuation of application Ser. No. 08/916,751, filed on Aug.19, 1997 now U.S. Pat. No. 5,916,426, which is a continuation of Ser.No. 08/637,057, filed on Apr. 24, 1996, abandoned; which is acontinuation of Ser. No. 08/458,525, filed on Jun. 2, 1995 and issued asU.S. Pat. No. 5,552,028 on Sep. 3, 1996; which is a divisional of Ser.No. 08/350,852, filed on Dec. 6, 1994 and issued as U.S. Pat. No.5,567,292 on Oct. 22, 1996; which is a continuation-in-part of Ser. No.08/170,078, filed on Dec. 17, 1993, now abandoned, which applicationsare incorporated herein by reference and to which applications priorityis claimed under 35 USC §120.

FIELD OF THE INVENTION

The invention relates generally to the field of capillaryelectrophoresis, and more particularly to materials and methods forsuppressing electroendoosmotic flow and analyte-wall interactions duringseparation of biomolecules, especially polynucleotides, by capillaryelectrophoresis.

BACKGROUND

Capillary electrophoresis has been applied widely as an analyticaltechnique because of several technical advantages: (i) capillaries havehigh surface-to-volume ratios which permit more efficient heatdissipation which, in turn, permit high electric fields to be used formore rapid separations; (ii) the technique requires minimal samplevolumes; (iii) superior resolution of most analytes is attainable; and(iv) the technique is amenable to automation, e.g. Camilleri, editor,Capillary Electrophoresis: Theory and Practice (CRC Press, Boca Raton,1993); and Grossman et al, editors, Capillary Electrophoresis (AcademicPress, San Diego, 1992). Because of these advantages, there has beengreat interest in applying capillary electrophoresis to the separationof biomolecules, particularly in nucleic acid analysis. The need forrapid and accurate separation of nucleic acids, particularlydeoxyribonucleic acid (DNA) arises in the analysis of polymerase chainreaction (PCR) products and DNA sequencing fragment analysis, e.g.Williams, Methods 4: 227-232 (19920; Drossman et al, Anal. Chem., 62:900-903 (1990); Huang et al, Anal. Chem., 64: 2149-2154 (1992); andSwerdlow et al, Nucleic Acids Research, 18: 1415-1419 (1990).

Since the charge-to-frictional drag ratio is the same for differentsized polynucleotides in free solution, electrophoretic separationrequires the presence of a sieving medium. The initial sieving media ofchoice were gels, but problems of stability and manufacturability haveled to the examination of non-gel liquid polymeric sieving media, suchas linear polyacrylamide, hydroxyalkylcellulose, agarose, and celluloseacetate, and the like, e.g. Bode, Anal. Biochem., 83: 204-210 (1977);Bode, Anal. Biochem., 83: 364-371 (1977); Bode, Anal. Biochem., 92:99-110 (1979); Hjerten et al, J. Liquid Chromatography, 12: 2471-2477(1989); Grossman, U.S. Pat. No. 5,126,021; Zhu et al, U.S. Pat. No.5,089,111; Tietz et al, Electrophoresis, 13: 614-616 (1992).

Another factor that complicates separations by capillary electrophoresisis the phenomena of electroendoosmosis. This phenomena, sometimesreferred to as electroosmosis, is fluid flow in a capillary induced byan electrical field. It has impeded the application of capillaryelectrophoresis to situations where high resolution separations arerequired, such as in the analysis of DNA sequencing fragments. Thephenomena arises in capillary electrophoresis when the inner wall of thecapillary contains immobilized charges which cause the formation of amobile layer of counter ions which, in turn, moves in the presence of anelectrical field to create a bulk flow of liquid. Unfortunately, themagnitude of the electroendoosmotic flow can vary depending on a host offactors, including variation in the distribution of charges, selectiveadsorption of components of the analyte and/or separation medium, pH ofthe separation medium, and the like. Because this variability tends toreduce ones ability to resolve closely spaced bands analyte, manyattempts have been made to directly or indirectly control such flow. Theattempts have included covalent modification of the inner wall of thecapillary to suppress charged groups, use of high viscosity polymers,adjustment of buffer pH and/or concentration, use of a gel separationmedium covalently attached to the capillary wall, and the application ofan electric field radial to the axis of the capillary, e.g. Hayes et al,Anal. Chem., 65: 2010-2013 (1993); Drossman et al (cited above);Hjerten, U.S. Pat. No. 4,680,201; Van Alstine et al, U.S. Pat. No.4,690,749; Wiktorowicz et al, Electrophoresis, 11: 769-773 (1990);Belder et al, J. High Resolution Chromatography, 15: 686-693 (1992).

Most of these approaches have met with mixed success or have only beenused in the separation of analytes quite different chemically fromnucleic acids. In particular, the use of capillary gels for DNAseparations have been hampered by manufacturing problems and problems ofstability and reliability during use, e.g. Swerdlow et al,Electrophoresis, 13: 475-483 (1992).

In view of the strong scientific and industrial interest in being ableto conveniently and accurately separate a variety of biomolecules,particularly polynucleotides, it would be desirable to have available alow viscosity electrophoretic separation medium capable of suppressingelectroendoosmotic flow and of reducing analyte-wall interactions.

SUMMARY OF THE INVENTION

The invention relates to the use of uncharged water-solublesilica-adsorbing polymers to suppress electroendoosmotic flow and toreduce analyte-wall interactions in capillary electrophoresis. In oneaspect of the invention, one or more of such polymers are employed ascomponents of a separation medium for the separation of biomolecules,preferably polynucleotides, by capillary electrophoresis. Generally,such polymers are characterized by (i) water solubility over thetemperature range between about 20° C. to about 50° C., (ii)concentration in a separation medium in the range between about 0.001%to about 10% (weight/volume), (iii) molecular weight in the range ofabout 5×10³ to about 1×10⁶ daltons, and (iv) absence of charged groupsin an aqueous medium having pH in the range of about 6 to about 9.Preferably, such polymers of the invention are substantiallynon-hydroxylic. In one embodiment, polymers of the invention areselected from the group consisting of polyvinylactams, such aspolyvinylpyrrolidone; N,N-disubstituted polyacrylamides; andN-substituted polyacrylamides. More preferably, such polymers of theinvention are poly(N,N-dimethylacrylamide).

In accordance with the method of the invention, a sufficient amount ofpolymer adsorbs to the silica surface to establish a zone of highviscosity at the silica surface that impedes the movement of anelectrical double layer under an electric field and that shields theanalyte from the wall.

The invention includes methods of using the polymers of the invention toseparate biomolecules, especially polynucleotides, by capillaryelectrophoresis; compositions comprising polymers of the invention forelectrophoretically separating biomolecules in capillaries; and methodsof using the separation medium of the invention for sequencing DNA.

The invention enhances the precision of biomolecule separation byelectrophoresis in a capillary by dynamically suppressingelectroendoosmotic flow and wall-analyte interactions through theadsorption of the uncharged polymers of the invention onto the surfaceof the capillary. Suppression is dynamic in the sense that throughoutthe separation process polymers of the invention adsorb and desorb fromthe surface of a capillary in equilibrium with polymer in solution inthe separation medium. Thus, a constant degree of suppression ismaintained not only during a separation run, but also from separationrun to separation run.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 diagrammatically illustrates an apparatus for carrying outcapillary electrophoresis.

FIG. 2 is an electropherogram of a 100 basepair DNA ladder separated ina 3% poly(dimethyacrylamide) solution (RM8) in a glycylglycine buffer.

FIG. 3 is an electropherogram of a 100 basepair DNA ladder separated ina 3% poly(dimethyacrylamide) solution (RM18) in a glycylglycine buffer.

FIG. 4 is an electropherogram of a 100 basepair DNA ladder separated ina 3% poly(dimethyacrylamide) solution (RM 18) in a TBE buffer.

FIG. 5 is an electropherogram of a 100 basepair DNA ladder separated ina binary polymer solution comprising 3% polyacrylamide and 0.05%poly(dimethylacrylamide) (RM 18) in a glycylglycine buffer.

FIGS. 6A to 6F are electropherograms of a 100 basepair DNA ladderseparated in various binary polymer solutions.

FIGS. 7A to 7J is an electropherogram of a commercially available DNAsequencing fragment standard separated in a separation medium containinga 6.5% solution of poly(dimethylacrylamide).

FIGS. 8A to 8F is an electropherogram showing the separation andsequencing of a 4-color sequencing standard in a separation mediumcontaining a 6.5% solution of poly(dimethylacrylamide). The numbersabove the peaks refer to the base number in the sequence, and theletters above each peak refer to the identity of the base.

FIG. 9 is an electropherogram showing a 4-color DNA sequencing analysisin polyvinylpyrrolidone by capillary electrophoresis.

FIGS. 9A to 9F are an electropherogram showing the separation andsequencing of a 4-color sequencing standard in a separation mediumcontaining a 10% solution of polyvinylpyrrolidone. The numbers above thepeaks refer to the base number in the sequence, and the letters aboveeach peak refer to the idenity of the base.

DEFINITIONS

The term “capillary” as used herein refers to a tube or channel or otherstructure capable of supporting a volume of separation medium forcarrying out electrophoresis. The geometry of a capillary may varywidely and includes tubes with circular, rectangular or squarecross-sections, channels, groves, plates, and the like, and may befabricated by a wide range of technologies. An important feature of acapillary for use with the invention is the surface-to-volume ratio ofthe surface in contact with the volume of separation medium. High valuesof this ratio permit better heat transfer from the separation mediumduring electrophoresis. Preferably, values in the range of about 0.4 to0.04 are employed. These correspond to the surface-to-volume ratios oftubular capillaries with circular cross-sections having inside diametersin the range of about 10 μm to about 100 μm. Preferably, capillaries foruse with the invention are made of silica, fused silica, quartz,silicate-based glass, such as borosilicate glass, phosphate glass,alumina-containing glass, and the like, or other silica-like materials.

The term “biomolecule” means a molecule typically synthesized by abiological organism that is water soluble and charged in the pH range offrom about 6 to about 9. Preferably, the term biomolecule includesproteins, glycoproteins, natural and synthetic peptides, alkaloids,polysaccharides, polynucleotides, and the like. More preferably, theterm biomolecule refers to polynucleotides.

The term “polynucleotide” as used herein refers to linear polymers ofnatural or modified nucleoside monomers, including double and singlestranded deoxyribonucleosides, ribonucleosides, α-anomeric formsthereof, and the like. Usually the nucleoside monomers are linked byphosphodiester bonds or analogs thereof to form polynucleotides rangingin size from a few monomeric units, e.g. 8-40, to several thousands ofmonomeric units. Whenever a polynucleotide is represented by a sequenceof letters, such as “ATGCCTG,” it will be understood that thenucleotides are in 5′→3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes thymidine, unless otherwise noted. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2nd Ed. (Freemán, San Francisco, 1992). “Analogs” inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described generallyby Scheit, Nucleotide Analogs (John Wiley, New York, 1980).

The term “electroendoosmosis” or “electroendoosmostic flow” as usedherein refers to the bulk flow of liquid due to the influence of anelectric field on the layer of mobile counter ions adjacent to fixed, orimmobile, charges on a surface, such as a capillary wall.Electroendoosmotic flow is typically measured as the mobility(cm²/sec-volts) of a test analyte through a capillary tube under astandard set of conditions, e.g. determining buffer concentration andtype, tube length, electrical field strength, and the like.

The term “polymer” is a large molecule composed of smaller monomericsubunits covalently linked together in a characteristic fashion. A“homopolymer” is a polymer made up of only one kind of monomericsubunit. A “copolymer” refers to a polymer made up of two or more kindsof monomeric subunits. As used herein the term “polymer” includeshomopolymers and copolymers. A “monodisperse” polymer solution meansthat the polymer molecules in solution have substantially identicalmolecular weights. A “polydisperse” polymer solution means that thepolymer molecules in solution have a distribution of molecular weights.

The term “non-hydroxylic” as used herein in reference to polymers meansthat the monomers used in the synthesis of a polymer contain no hydroxylsubstituents.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a convenient means for suppressingelectroendoosmotic flow and wall-analyte interactions during theseparation of biomolecules, particularly DNA, by capillaryelectrophoresis. As used herein, the term “separation medium” refers tothe medium in a capillary in which the separation of analyte componentstakes place. Separation media typically comprise several components, atleast one of which is a charge-carrying component, or electrolyte. Thecharge-carrying component is usually part of a buffer system formaintaining the separation medium at a constant pH. Media for separatingpolynucleotides, or other biomolecules having different sizes butidentical charge-frictional drag ratios in free solution, furtherinclude a sieving component. In addition to such conventionalcomponents, the separation medium of the invention comprise a surfaceinteraction component. In the case of polynucleotide separations, thesieving component may be the same or different than the surfaceinteraction component, but is usually different. The surface interactioncomponent comprises one or more uncharged water-soluble silica-adsorbingpolymers having the physical properties set forth above. Preferably,such one or more uncharged water-soluble silica-adsorbing polymers arenon-hydroxylic. In further preference for polynucleotide separations,the sieving component of the separation medium of the inventioncomprises one or more uncrosslinked, particularly linear, polymers.Preferably, the components of the separation medium of the invention areselected so that its viscosity is low enough to permit rapid re-fillingof capillaries between separation runs. For typical capillaries, e.g.20-100 μm inside diameter and 40-60 cm in length, in the absence of asieving component, viscosity is preferably less than 1000 centipoise,and more preferably, between about 1 to about 300 centipoise. In thepresence of a sieving component, viscosity is preferably less than 5000centipoise, and more preferably, less than 1000 centipoise.

Polymers for use as the surface interaction component of the separationmedium may belong to a variety of chemical classes, such as thosedescribed in the following references: Molyneux, Water-Soluble SyntheticPolymers: Properties and Behavior, Volumes I and II (CRC Press, BocaRaton, 1982); Davidson, Editor, Handbook of Water-Soluble Gums andResins (McGraw-Hill, New York, 1980); Franks, editor, Water: AComprehensive Treatise (Plenum Press, New York, 1973); and the like.Preferably, the uncharged water-soluble silica-adsorbing polymers of theinvention include, but not limited to, N,N-disubstitutedpolyacrylamides, N-monosubstituted polyacrylamides, polymethacrylamide,polyvinylpyrrolidone, and the like. Exemplary substituents of thepolyacrylamides include C₁ to C₁₂ alky; halo-substituted C₁ to C₁₂alkyl; methoxy-substituted C₁ to C₁₂ alkyl; hydroxyl-substituted C₁ toC₁₂ alkyl and the like. Preferably, the halo substituent is fluoro andthe hydroxyl-substituted C₁ to C₁₂ alkyl is monosubstituted. It isunderstood that the above monomer substituents are selected so that theresulting polymer is water soluble. For example, it is clear that C₁₂alkyl-containing mononer could only be present as a small fractionalcomponent of a copolymer. More preferably, exemplary substituents areselected from the group consisting of C₁ to C₃ alkyl; halo-substitutedC₁ to C₃ alkyl; methoxy-substituted C₁ to C₃ alkyl; andhydroxyl-substituted C₁ to C₃ alkyl.

Such polymers are synthesized by conventional techniques, e.g. asdisclosed in Odian, Principles of Polymerization, Third Edition (JohnWiley, New York, 1991). An important feature of the invention is thatthe polymer of the surface interaction component be uncharged.Preferably, polymers of the invention are synthesized under non-aqueousconditions so that uncharged initiators can be used. Such conditionsalso preclude the incorporation of charged initiators into the product.The polymers comprising the surface interaction component of theseparation medium may be present at a concentration of from about 0.001%to about 10% (w:v). Preferably, such polymers are present at aconcentration in the range of about 0.01% to about 6%.

The silica-adsorbing quality of the preferred polymers can be measuredin a number of well-known ways, such as by ellipsometry, determiningchanges in the hydrodynamic properties of adsorbent test particles,determination of adsorption isotherms, or like methods. Such techniquesare described in Malmsten et al, Macromolecules, 25: 2474-2481 (1992);Rob and Smith, European Polymer J., 10: 1005-1010 (1974); Vincent et alSurf. Colloid Sci., 12: 1-117 (1982); Takahashi et al, Advances inPolymers Science, 46: 1-65 (1982), and like references. An adsorptionisotherm is a graphical presentation of the adsorption exerted by anadsorbent on a solution of a given substance at a fixed temperature. Thedetermination of adsorption isotherms require the preparation ofsolutions of known concentrations of the material whose adsorption is tobe measured (the adsorbate). The adsorbate solutions are combined withknown quantities of the material (the adsorbent) whose surface theadsorbate adheres to. Once an equilibrium is reached between theadsorbate in solution and the adsorbate on the surface of the adsorbent,the concentration of the adsorbate solution is determined. The reductionin concentration of the solution is a measure of the degree ofadsorption of the adsorbate under the standard conditions.

The degree of adsorption may also be measured indirectly by observingthe reduction of electroendoosmotic flow under a set of standard valuesof the following parameters: buffer type and concentration, temperature,electric field strength, capillary type, diameter, and length, and testanalyte. An exemplary standard for such measurement is as follows:Uncoated fused silica capillary 40 cm in total length, 20 cm to detector(UV), 75 μm inside diameter; 0.1 M glycylglycine buffer (pH 8.0); markersolution of 0.92 mM mesityl oxide and 1 mM p-toluenesulfonic acid(p-TSA); electrophoresis at 30° C. under 10 kV. The polymer being testedis added to the buffer. With no surface interaction component, theelectroendoosmotic flow is approximately 6×10⁻⁴ cm²/sec-volts.Preferably, in such a separation medium, a sufficient concentration ofpolymer of the invention is employed to reduce electroendoosmotic flowto less than about 2×10⁻⁵ cm²/sec-volts.

For polynucleotide separations, the silica-adsorbing quality of apolymer of the invention is preferably characterized by the relationshipbetween resolving power and polynucleotide length for a selected“ladder” of polynucleotides under a standard set of conditions.Resolving power is conveniently expressed in terms of the number oftheoretical plates, N, of the test system. N=(L/σ)² where L is theaverage path length of a test analyte under a peak from injection portto detector (usually position of peak maximum) and σ is the variance ofthe peak. Preferably, polymers of the invention provide a substantiallylinear relationship between number of theoretical plates and size ofpolynucleotide over the range of from about 100 to about 500nucleotides; more preferably, the relationship is linear over the rangeof from about 20 to about 600 nucleotides. A standard set of conditionsfor generating theoretical plates versus polynucleotide length curves isdescribed below.

Exemplary ladders of different-sized polynucleotides in theabove-mentioned size ranges are available in commercially availablekits, e.g. the 100 basepair double stranded DNA ladder from BRL-GIBCO,the Taq DNA Sequencing Standard from Applied Biosystems, Inc., or thelike. A standard separation medium can be prepared as follows: 0.60 g ofacrylamide (ultrapure, ICN, Costa Mesa, Calif.) is dissolved in 10 ml1×TBE, 30% formamide, 3.5 M urea buffer, filtered (0.2 μm pore size),and degassed. The monomer solutions are polymerized by addition at roomtemperature of 1 μl of 100% N,N,N′,N′-tetramethylethylenediamine (TEMED)and 2 μl ammonium persulfate, 10% w:v in water (APS), per ml of monomersolution (to give a final concentration of 0.02% w:v APS and 0.1% v:vTEMED).

The above separation medium is loaded into a 55 cm uncoated fused silicacapillary tube, 50 μm inside diameter, 40 cm to detector. The capillarymay be used in a commercially available capillary electrophoresisapparatus having fluorescence detection capability. Fluorescencedetection systems for detecting fluorescently labelled analytes incapillaries is well known in the art, e.g. Mathies et al, U.S. Pat. No.5,091,652; Mathies et al, International Application No. PCT/US93/01607;Ruiz-Martinez et al, Anal. Chem. 65: 2851-2858 (1993); and the like. TheDNA fragments from the standard are denatured and loadedelectrokinetically as follows: The dried sample is resuspended in amixture of 5 mM aqueous EDTA (0.5 μl) and formamide (6 μl). Thesuspension is heated at 90° C. for 2 minutes then transferred to an icebath. The ladder is loaded by placing the cathode and cathodic end ofthe capillary into the above solution then applying 6 kV across the tubefor 5 seconds. Separation of the DNA fragments in the ladder commencesby returning the cathode and cathodic end of the capillary into thecathode reservoir and applying a running voltage of 12 kV.

Apparatus for carrying out capillary electrophoresis is well-known andis not a critical feature of the invention. Many references areavailable describing the basic apparatus and several capillaryelectrophoresis instruments are commercially available, e.g. AppliedBiosystems (Foster City, Calif.) model 270A instrument. Exemplaryreferences describing capillary electrophoresis apparatus and theiroperation include Jorgenson, Methods, 4: 179-190 (1992); Colburn et al,Applied Biosystems Research News, issue 1 (winter 1990); Grossman et al(cited above); and the like. FIG. 1 is a schematic representation of anexemplary capillary electrophoresis system 20 suitable for practicingthe invention. However, as mentioned above, a wide variety of systemsare amenable for use with the invention in addition to that representedin the figure, e.g. as described in Harrison et al, Science, 261:895-897 (1993); Pace, U.S. Pat. No. 4,908,112; Kambara et al, U.S. Pat.No. 5,192,412; Seiler et al, Anal. Chem., 65: 1481-1488 (1993); and thelike. In the figure, capillary tube 22 preferably has a length betweenabout 10 to 200 cm, typically less than about 100 cm, and a preferredinner diameter in the range of about 10 to 200 μm, and more typically inthe range of about 50 to 75 μm, e.g. available from PolymicroTechnologies (Phoeniz, Ariz.). Preferably, there is no coating on theinside surface of the tube. A cathodic reservoir 26 in system 20contains a separation medium 28, described further below. The cathodicend 22 a of capillary tube 22 is sealed within reservoir 26 and isimmersed in the separation medium during electrophoresis. Second tube 30in reservoir 26 is connected to a finely controlled air pressure systemwhich can be used to control the pressure in the head space above theseparation medium, e.g. for loading separation medium into the capillarytube by positive pressure. Sample reservoir 31 contains the samplemixture to be loaded into the cathodic end of capillary 22. The anodicend 22 b of capillary 22 is immersed in separation medium 32 containedin anodic reservoir 34. A second tube 36 in reservoir 34 can be includedto control the pressure above separation medium 32. High voltage supply40 is connected to the cathodic and anodic reservoirs by electrodes 41and 42. High voltage supply 40 produces a constant potential across theelectrodes in the range of a few kilovolts (kV) to 60 kV, with apotential in the range of about 10 to 30 kV being typical. Currentsthrough the capillary are generally in the microamp range, typicallybetween a few to 100 μA, with 20 μA being typical. Detector 44positioned adjacent to capillary 22 monitors sample peaks migratingthrough optical detection zone 45 of the capillary. Typically, opticaldetection zone 45 comprises a region of capillary 22 in which the ususalpolyimide coating has been removed to permit UV and/or visible light,e.g. fluorescence, detection of the separated analyte. A wide variety ofdetection schemes are amenable for use with the invention, including UVabsorption, fluorescence emission, conductance, radioactive emission,and the like. For example, detection systems for fluorescent analytesare described in Zare et al, U.S. Pat. No. 4,675,300 and Folestad et al,U.S. Pat. No. 4,548,498.

As mentioned above, separation medium of the invention generallycomprises three components: a charge-carrying component, a sievingcomponent, and a surface interaction component. Additional componentsmay also be included in particular embodiments, such as denaturants whenit is desirable to prevent the formation of duplexes or secondarystructures in polynucleotides. Preferred denaturants include formamide,e.g. 40-90%, urea, e.g. 6-8 M, commercially available lactams, such aspyrrolidone, and the like. Guidance for their use in electrophoresis canbe found in well known molecular biology references, e.g. Sambrook etal, Molecular Cloning: A Laboratory Manual, Second Edition (Cold SpringHarbor Laboratory, New York, 1989).

Typically, a buffer system for controlling pH is employed as thecharge-carrying component. Exemplary buffers include aqueous solutionsof organic acids, such as citric, acetic, or formic acid; zwitterionics,such as TES (N-tris[hydroxymethyl]-2-aminoethanesulfonic acid, BICINE(N,N-bis[2-hydroxyethyl]glycine, ACES(2-[2-amino-2-oxoethyl)-amino]ethanesulfonic acid), or glycylglycine;inorganic acids, such as phosphoric; and organic bases, such as Tris(Tris[hydroxymethyl]aminomethane) buffers, e.g. available from Sigma.Buffer concentration can vary widely, for example between about 1 mM to1 M, but are typically about 20 mM. Exemplary buffer solutions forconventional capillary electrophoresis applications include thefollowing: (i) 0.1 M Tris, 0.25 M boric acid, 7 M urea with a pH of 7.6for single stranded polynucleotide separations; or (ii) 0.089 M Tris,0.089 M boric acid, 0.005 M EDTA for double stranded polynucleotideseparations. For non-zwitterionic buffer systems, preferably PDMA orpolyvinylpyrrolidone are employed as the surface interaction component.

Sieving components of electrophoretic separation media are well known inthe art and are disclosed in Zhu et al, U.S. Pat. No. 5,089,111;Ruiz-Martinez et al, Anal. Chem., 65: 2851-2858 (1993); Williams,Methods, 4: 227-232 (1992); and like references. Preferably, the sievingcomponent of the separation medium of the invention is a low-viscosityentangled polymer solution as taught by Grossman, U.S. Pat. No.5,126,021. A low viscosity separation medium is preferred so thatcapillaries can be readily re-filled in automated systems, e.g. forlarge-scale DNA sequencing applications. The rate of solution flowthrough the capillary determines how much time is required to replacethe separation medium between successive analyses. Guidance forsynthesizing entangled polymers with a range of viscosities suitable forDNA sieving applications is provided by Grossman, which is incorporatedby reference. Generally, the viscosity of a polymer, or copolymer,solution is determined by the molecular weight (MW) and concentration ofthe polymer or copolymer components of the separation medium. Themolecular weight of a polymer or copolymer can be adjusted duringsynthesis in a number of ways well known in the art, e.g. as reviewed inOdian, Principles of Polymerization, Third Edition (John Wiley, NewYork, 1991), or like references.

A second approach for controlling the average MW of a polymer orcopolymer used in the invention is by fractionating a polydispersepolymer product into different MW fractions followed by purification.Typical fractionation techniques include gel permeation chromatography,dialysis using membranes having specific MW cutoffs, fractionalprecipitations in water-miscible solvents, such as methanol, and thelike.

For apparatus employing conventional capillary tubes, it is clear thatthe upper limits of polymer or copolymer MW and/or concentration isdictated primarily by the upper viscosity that can be pushed or pulledthrough the tubes. For example, if short capillaries (length of about 20cm) with large inside diameters (IDs) (e.g. radius of about 0.01 cm) areemployed, a solution with a viscosity of as much as 38,000 centipoisecould be pushed through the capillary in 30 minutes at high pressure,e.g. 100 psi. For more conventional capillary tubes, e.g. 50 μm ID and50 cm in length, a viscosity in the range of about 10-1000 centipoisepermits separation medium to be replaced within about 30 minutes using apressure differential across the tube of between about 50-100 psi.

Exemplary sieving polymers include linear polyoxides; polyethers, suchas polyethylene oxide and polypropylene oxide; polyacrylamide;polymethacrylamide; polyvinylpyrrolidone; polyvinyloxazolidone; and avariety of water-soluble hydroxylic polymers, such as water-solublenatural gums, such as dextran; water-soluble cellulose compounds, suchas methylcellulose and hydroxyethylcellulose, and copolymers and blendsof these polymers. Preferably, such polymers are used at a concentrationin the range between about 0.5% and 10% w:v.

Double stranded polynucleotides, e.g. DNA fragments from PCR or LCRamplifications, enzyme digests, or the like, are separated by standardprotocols, or manufacturer's suggested protocols where a commercialcapillary electrophoresis instrument is employed, e.g. a model 270-HTinstrument (Applied Biosystems, Inc., Foster City). The only exceptionto such standard or suggested protocols is that the separation medium ofthe invention is employed.

DNA sequencing in accordance with the invention requires the separationof single stranded polynucleotides prepared by DNA sequencing protocols,e.g. described in Sambrook et al, Molecular Cloning: A LaboratoryManual, Second Edition (Cold Spring Harbor Laboratory, New York, 1989);Ausubel et al, Current Protocols in Molecular Biology (John Wiley &Sons, Media, PA); or the like.

The important feature of currently available DNA sequencing protocols isthe generation of a “nested series” or “ladder” of single strandedpolynucleotides, or DNA sequencing fragment, that must be separated bysize. The basic steps of the chain-termination approach to DNAsequencing are (1) providing an oligonucleotide primer and a templatenucleic acid containing, as a subsequence, a target nucleic acid whosesequence is to be determined, (2) hybridizing the oligonucleotide primerto the template nucleic acid, (3) extending the primer with a nucleicacid polymerase, e.g. T7 DNA polymerase, Sequenase™, a reversetranscriptase, or the like, in a reaction mixture containing nucleosidetriphosphate precursors and at least one chain terminating nucleotide toform a nested series of DNA fragment populations, such that everyshorter DNA fragment is a subsequence of every longer DNA fragment andsuch that each DNA fragment of the same size terminates with the samechain-terminating nucleotide, (4) separating the DNA fragmentpopulations according to size, and (5) identifying the chain-terminatingnucleotide associated with each DNA fragment population.

As used herein, the term “nucleoside triphosphate precursors” refers todeoxyadenosine triphosphate (ATP), deoxycytidine triphosphate (CTP),deoxyguanosine triphosphate (GTP), and thymidine triphosphate (TTP), oranalogs thereof, such as deoxyinosine triphosphate (ITP),7-deazadeoxyguanosine triphosphate, and the like.

A template is provided in accordance with the teachings in the art, e.g.Technical Manual for Model 370A DNA Sequencer (Applied Biosystems, Inc.,Foster City, Calif.). For example, the target sequence may be insertedinto a suitable cloning vector, such as the replicative form of an M13cloning vector, which is then propagated to amplify the number of copiesof the target sequence. The single-stranded form of M13 is isolated foruse as a template. Alternatively, a template can be provided bypolymerase chain reaction (PCR) as taught in the art, e.g. Innis et al,(cited above); Wilson et al, Biotechniques, Vol. 8, pgs. 184-189 (1990);Gyllensten, Biotechniques, Vol. 7, pgs. 700-708 (1989); and the like.After amplification, the template can be used in the polymerizationreaction(s) either in liquid phase or attached to a solid phase support,e.g. as taught by Stahl et al, Nucleic Acids Research, Vol. 16, pgs.3025-3038 (1988); Hultman et al, Nucleic Acids Research, Vol. 17, pgs.4937-4946 (1989); or the like.

Once the nested series DNA fragments are generated, they are separatedby capillary electrophoresis using the separation medium of theinvention.

EXAMPLE 1 Synthesis of PDMA in Dioxane Using AIBN

Poly(N,N-dimethylacrylamide) (pDMA) is synthesized using conventionaltechniques, e.g. as disclosed in Trossarelli et al, J. Polymer Sci.,57:445-452 (1962). Known amounts of dimethylacrylamide (DMA), dioxane,and azobisisobutyronitrile (AIBN) were mixed in an Erlenmeyer flask andargon gas was bubbled through the solution for 10 minutes at roomtemperature. Polymerization was initiated by raising the temperature to55° C. Polymerization times ranged from 10 to 25 minutes depending onthe concentration of monomer. After polymerization, the resultingpolymer was purified by three cycles of precipitation in hexane anddissolution in CH₂Cl₂. Finally, the hexane precipitate was driedovernight in a vacuum desiccator then lyophilized. The table belowsummarizes the reaction conditions for the various experiments.

Estimated Concentration Monomer Molecular Average Batch No. (% w/v)Dioxane (cc) AIBN (mg) Weight* RM1 70 14.3 12 79 kd RM2 60 17.0 14 92 kdRM3 50 20.0 16 99 kd RM4 40 25.0 21 97 kd RM5 30 33.3 27 83 kd RM6 2050.0 41 — RM7 10 100.0 82 69 kd RM8  5 200.0 164  54 kd *Estimated bygel permeation chromatography (peak mol. wt.).

EXAMPLE 2 Synthesis of PDMA in t-butyl Alcohol Using AIBN

Further polymerizations were carried out with t-butyl alcohol (t-BuOH)using the following protocol: Known amounts of DMA monomer, t-butylalcohol, and AIBN were combined, and argon gas was bubbled through thesolutions for 20 minutes. The mixtures were brought to 55° C. andallowed to polymerize for 15 minutes. The resulting polymers wereisolated as described in Example 1. The table below summarizes thereaction conditions for the various experiments.

Estimated Monomer Average Batch Concentration AIBN Monomer Molecular No.(% w/v) t-BuOH (cc) (mg) (g) Weight RM17 50 20.0 16 10 81 kd RM18 5060.0 50 30 107 kd  RM19 70 14.0 12 10 99 kd RM21 70 72.0 60 50 112 kd *Estimated by gel permeation chromatography (peak mol. wt.).

EXAMPLE 3 Change in Electroendoosmotic Flow in Test System by VariousPoly(dimethylacrylamide) Solutions

The effect of various formulations of PDMA on electroendoosmotic flow ina test system was measured. The test system consisted of an AppliedBiosystems model 270 HT capillary electrophoresis instrument configuredin the following manner: Uncoated fused silica capillary 40 cm in totallength, 20 cm to detector (UV), 75 μm inside diameter was installed; theseparation medium consisted of a 0.1 M glycylglycine buffer (pH 8.0)with the test PDMA polymer added; a marker solution consisted of 0.92 mMmesityl oxide; and electrophoresis took place at 30° C. under 10 kVafter electrokinetic loading as described above. The results are listedin the table below:

Electroendo- osmotic PDMA Concentration Flow* RM8 0.1% (w:v) 7.38 × 10⁻⁵RM16 0.1% (w:v) 2.73 × 10⁻⁵ RM18** 0.01% (w:v)  1.98 × 10⁻⁵*cm²/sec-volts **p-TSA (1 mM in H₂O) used as marker.

EXAMPLE 4 Electrophoresis of 100 Basepair DNA Ladder Using 3% RM8 in a0.1 M Glycylglycine Buffer

A 3% (w:v) RM8 PDMA polymer in a 0.1 M glycylglycine buffer was used toseparate the components of a commercial double stranded DNA ladder (100bp DNA ladder, GIBCO-BRL). An Applied Biosystems model 270 HT was fittedwith a 75 μm inside diameter uncoated fused silica capillary having 60cm total length and 40 cm from the sample injection port to detector.Electrophoresis was carried out under 10 kV and 13 μA at 30° C. Thesample was electrokinetically injected under 5 kV and 6 μA for 5seconds. An electropherogram of the analyte (showing UV absorption at260 nm) is illustrated in FIG. 2.

EXAMPLE 5 Electrophoresis of 100 Basepair DNA Ladder Using 3% RM18 in a0.1 M Glycylglycine Buffer

A 3% (w:v) RM18 PDMA polymer in a 0.1 M glycylglycine buffer pH 8.0 wasused to separate the components of the double stranded DNA ladder ofExample 4. An Applied Biosystems model 270 HT was fitted with a 75 μminside diameter uncoated fused silica capillary having 60 cm totallength and 40 cm from the sample injection port to detector.Electrophoresis was carried out under 10 kV and 13 μA at 30° C. Thesample was electrokinetically injected under 5 kV and 7 μA for 5seconds. An electropherogram of the analyte (showing UV absorption at260 nm) is illustrated in FIG. 3.

EXAMPLE 6 Electrophoresis of 100 Basepair DNA Ladder Using 3% RM18 in a90 mM TBE Buffer

A 3% (w:v) RM18 PDMA polymer in a 90 mM TBE buffer pH 8.3 was used toseparate the components of the double stranded DNA ladder of Example 4.An Applied Biosystems model 270 HT was fitted with a 75 μm insidediameter uncoated fused silica capillary having 60 cm total length and40 cm from the sample injection port to detector. Electrophoresis wascarried out under 10 kV and 8 μA at 30° C. The sample waselectrokinetically injected under 5 kV and 8 μA for 5 seconds. Anelectropherogram of the analyte (showing UV absorption at 260 nm) isillustrated in FIG. 4.

EXAMPLE 7 Electrophoresis of 100 Basepair DNA Ladder Using 3%Polyacrylamide With and Without 0.05% PDMA (RM18) in a 0.1 GlycylglycineBuffer

A 3% (w:v) linear polyacrylamide solution in a 0.1 M glycylglycinebuffer pH 8.0 was used to separate the components of the double strandedDNA ladder of Example 4. An Applied Biosystems model 270 HT was fittedwith a 75 μm inside diameter uncoated fused silica capillary having 60cm total length and 40 cm from the sample injection port to detector.Electrophoresis was carried out under 10 kV and 17 μA at 30° C. Thesample was electrokinetically injected under 5 kV and 8 μA for 5seconds. After 30 minutes no peaks were detected indicating that therewas no separation of ladder components.

A second separation was conducted under identical conditions, exceptthat the polymer solution used was a mixture of 3% linear polyacrylamideand 0.05% PDMA (RM18). An electropherogram of the analyte (showing UVabsorption at 260 nm) is illustrated in FIG. 5.

EXAMPLE 8 Electrophoresis of 100 Basepair DNA Ladder Using PolymerSolutions of Polyethylene oxide and Poly-N-vinvlpyrrolidone in 0.1 MGlycylglycine Buffers

A 3% (w:v) solution of poly-N-vinylpyrrolidone (PVP) (average MW 360 kD)and a 5% (w:v) solution of polyethylene oxide (PEO) (average MW 35 kD)were prepared in 0.1 M glycylglycine buffers pH 8.0. The DNA ladder ofExample 4 was electrophoretically separated in six separate experimentsusing the same apparatus and under the same conditions as used inExamples 4-7 with the exception that different polymer solutions wereemployed. The polymer solutions are listed in the table below withreference to the Figures illustrating the degree of separationaccomplished. The poly(dimethylacrylamide) used was RM18.

Polymer Solution Separation Figure 3% PVP Yes 6A 6% PVP Yes 6B 3% PVP +0.5% PDMA Yes 6C 5% PEO No No figure 5% PEO + 0.5% PDMA Yes 6D 0.5% PDMAYes 6E 5% PEO + 0.05% PDMA Yes 6F

EXAMPLE 9 Separation of DNA Sequencing Fragments inPoly(dimethylacrylamide) by Capillary Electrophoresis

Fluorescently labelled DNA sequencing fragments were obtained fromApplied Biosystems, Inc. (Foster City, Calif.) (The fragments used werethe “C”-terminated fragments used to make up the 4-color sequencingstandard supplied by Applied Biosystems as Part No. 400993, Taq DNASequencing Standard). 8 μl of the mixture containing fragmentsterminating with dideoxycytidine and labelled with fluorescein (FAM-Cfragments) was added to a 500 μl centrifuge tube and dried in a speedvac using moderate heating. After adding 0.5 ml 50 mM EDTA solution and6 ml recrystalized formamide to the dried FAM-C fragments, the mixturewas heated at 95° C. for 2 min then placed on ice.

A separation medium for electrophoresis was prepared as follows: A stockbuffer was prepared by mixing 20 ml methanol 110 ml water and 2.8 g Trisfollowed by titration with 85% phosphoric acid to pH 8.0. The separationmedium was prepared by mixing 3.6 ml stock buffer, 3.6 ml water, 4.8 gurea, and 0.65 g poly(dimethylacrylamide) prepared as described above(RM21) to give a total volume of approximately 10 ml. The resultingmixture was stirred for 3 hours then filtered through a 0.45 μm syringefilter.

An uncoated 50 μm inside diameter Polymicro Technologies fused silicacapillary (Cat. No. 2000017) of total length 54 cm was prepared so thatthere was 40 cm between the injection inlet and the detection zone.Prior to the first use, the capillary was flushed with 20 column volumesof 1.0 M NaOH, 20 column volumes of water, then filled with separationmedium. In subsequent runs with the same capillary, prior to use, thecapillary was flushed with 20 column volumes of water, 20 column volumestetrahydrofuran (THF), 20 column volumes 1 M NaOH, 20 column volumes ofwater, then filled with separation medium.

The FAM-C fragment sample was electrokinetically loaded into thecapillary under 1.8 kV at 0.69 μA for 25 sec, taking care to keep theelectrode and the end of capillary as far apart as possible. Thefragments were separated under 220 V/cm at 4.41 μA. Both sampleinjection and electrophoresis took place at 22° C. Fragment bands wereilluminated at the detection window with an excitation beam from anargon ion laser (model 221-40MLA, Cyonics, San Jose, Calif.) operatingat 1.5 mW. The excitation beam was passed through a 0.5 optical densityneutral density filter (#FNG 085, Melles Groit, Irvine, Calif.) and intoa set of focusing optics composed of a 64 mm focal length 7 mm diameterpositive lens and an 85 mm focal length 5 mm diameter negative lens,resulting in a beam diameter of approximately 100 μm focused on thecapillary detection window. Fluorescence emission was collected at rightangles by a 12 mm focal length 14 diameter aspheric collector lens andpassed through a 530 nm RDF bandpass filter (Omega Optical, Brattleboro,Vt.) and to a Fabry set composed of a 48 mm focal length 19 mm diameteraspheric Fabry lens followed by a 17 mm 10 mm diameter spherical Fabrylens. The light was then imaged on a photomultiplier tube (#R98-21,Hamamatsu, San Jose, Calif.) for detection. The electropherogram of theseparated fragments is shown in FIGS. 7A-7J. The numbers adjacent to thepeaks indicate the fragment size.

EXAMPLE 10 4-Color DNA Sequencing Analysis in Poly(dimethylacrylamide)by Capillary Electrophoresis

Fluorescently labeled DNA sequencing fragments were obtained fromApplied Biosystems, Inc. (Foster City, Calif.) (Part No. 400993, Taq DNASequencing Standard). To the dry Sequencing Standard were added 30 μl ofa sample loading reagent made up of 0.15% hydroxyethylcellulose (QP100MHUnion Carbide) dissolved in a water-pyrrolidone (75:25 (vol:vol))solvent. The sample was then divided into two 15 μl aliquots, heated at95° C. for 2 min, and placed on ice.

The separation medium was prepared by dissolving 0.65 gpoly(dimethylacrylamide) prepared as described above (RM21) and 4.8 gurea in a solution of 1.0 ml 1.0 M TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid), pH 8.0, and6.2 ml water. The polymer solution was stirred overnight then filteredthrough a 0.45 μm syringe filter. The viscosity of the final polymersolution was approximately 75 cp at 25° C. as measured in a Brookfieldviscometer Model DV-II using spindle #00 at a speed of approximately 50rpm (Brookfield Engineering Laboratories, Stoughton, Mass.).

An uncoated 50 μm inside diameter fused silica capillary (PolymicroTechnologies, Tucson, Ariz. Cat. No. 2000017) of total length 51 cm wasprepared so that there was 40 cm between the injection inlet and thedetection zone. Prior to the first use, the capillary was flushed withgreater than 20 column volumes of water, followed by greater than 20column volumes of 0.1 M NaOH, followed by greater than 20 column volumesof water, then filled with separation medium.

The 4-color detection system used herein is similar to well knownsystems in the art of DNA analysis and is not a critical feature of thepresent invention, e.g., Karger et al., Nucleic Acids Research 19(18):4955-62 (1991). The 4-color detection system utilizes an argon ion laseras a fluorescence-excitation light source that emits light atwavelengths of 488 and 514 nm. Typically the laser was operated at atotal laser power of 9.9 mW. The laser light passes through a bandpassfilter to remove the laser tube's cathode glow, the filter passing lighthaving a wavelength of between approximately 485 nm and 515 nm. Next, aplano-convex lens diverges the light beam, the lens having a focallength of 100 mm and a diameter of 8 mm, e.g., Melles Griot part no.01LPK041/078 (Melles Griot, Irvine, Calif.). The laser light then passesthrough a dichroic mirror which passes light having wavelengths ofbetween approximately 485 nm and 515 nm, then passes through amicroscope objective and into the detection region of the separationcapillary. The emission light is reflected off of the dichroic mirrorand directed toward a spectrograph. To reduce the amount of scatteredlaser light passing onto the spectrograph, the emission light passesthrough a long-pass filter having a cutoff of approximately 520 nm andis then focused onto an entrance slit of the spectrograph by are-imaging lens having an 85 mm focal length, e.g., Melles Griot partno. 01LPK035. The spectrograph utilizes a 405 g/mm, 450 nm blaze gratingwith a dispersion of 17 nm/mm. After passing through the spectrograph,the light then falls onto a charged coupled device (CCD) detector. Theoutput signal from the CCD is transmitted to electronic computer forsubsequent data analysis and presentation. The software used for dataanalysis was the Sequencing Analysis version 2.1.0B1, which is similarto commercially utilized sequence analysis software (Applied BiosystemsModel 373 DNA Sequencer), the basic algorithm of which is generallydescribed elsewhere, e.g., Smith et al, Methods in Enzymology Vol. 155pages 260-301, Academic Press (1991).

The sample was electrokinetically loaded into the capillary using afield of 60 V/cm for 25 sec. The fragments were separated under a fieldof 160 V/cm at 3.0 μA at a temperature of 42° C. The run was allowed toproceed for approximately two hours.

The resulting electropherogram is shown in FIGS. 8A-8F.

EXAMPLE 11 4-Color DNA Sequencing Analysis in Polyvinylpyrrolidone byCapillary Electrophoresis

The separation medium was prepared by dissolving 1.0 gpolyvinylpyrrolidone (Povidone, United States Pharmacopia, BASF,Kollidon 90 F) and 4.8 g urea in a solution of 1.0 ml 1.0 M TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid), pH 8.0, and6.2 ml water. The polymer solution was stirred overnight then filteredthrough a 0.45 μm syringe filter.

The DNA sequencing fragments, the fragment sample preparation, theelectrophoresis capillary, the 4-color detection system, the sampleinjection protocol, and the electrophoresis run conditions were allessentially the same as those used in Example 10.

The resulting electropherogram is shown in FIGS. 9A—9F.

No dye mobility correction was applied to the data shown in FIG. 9.Because the addition of fluorescent dyes to the DNA sequencing extensionproducts alters the electrophoretic mobility of the asociated DNAfragments, and because different dyes cause different mobility shifts, a“mobility correction” is required to normalize the electrophoreticmobility of fragments containing different dyes. Because the data inFIG. 9 has not been corrected for these mobility shifts, the order ofthe peaks is offset somewhat. However, it is still possible to see thatthe requisite resolution of neighboring fragments has been achievedusing the polyvinylpyrrolidone material.

We claim:
 1. A composition for separating analytes by capillaryelectrophoresis, the composition comprising: a charge-carryingcomponent; a sieving component comprising an uncrosslinked polymer; anda surface interaction component comprising one or more polymers selectedfrom the group consisting of N,N-disubstituted polyacrylamide andN-substituted polyacrylamide, wherein said N substituents are selectedfrom the group consisting of C₁ to C₃ alkyl, halo-substituted C₁ to C₃alkyl, methoxy-substituted C₁ to C₃ alkyl, and hydroxyl-substituted C₁to C₃ alkyl; wherein the sieving component and the surface interactioncomponent are the same or different; and wherein the composition doesnot include a crosslinked polymer gel.
 2. The composition of claim 1wherein the composition has a viscosity of less than 5000 centipose at25° C.
 3. The composition of claim 1 wherein the surface interactioncomponent is poly(N,N-dimethylacrylamide).
 4. The composition of claim 1further including a denaturant.
 5. The composition of claim 4 whereinthe denaturant is selected from the group consisting of formamide, urea,and pyrrolidone.
 6. The composition of claim 5 wherein the denaturant ispyrrolidone.
 7. A composition for separating analytes by capillaryelectrophoresis, the composition comprising: a charge-carryingcomponent; a sieving component comprising an uncrosslinked polymer; anda surface interaction component comprising N,N-disubstitutedpolyacrylamide, wherein N substituents are selected from the groupconsisting of C₁ to C₃ alkyl, halo-substituted C₁ to C₃ alkyl,methoxy-substituted C₁ to C₃ alkyl, and hydroxyl-substituted C₁ to C₃alkyl; wherein the sieving component and the surface interactioncomponent are the same or different; and wherein the composition doesnot include a crosslinked polymer gel.
 8. A composition for separatinganalytes by capillary electrophoresis, the composition comprising: acharge-carrying component; a sieving component comprising anuncrosslinked polymer; and a surface interaction component comprisingN-substituted polyacrylamide, wherein N substituents are selected fromthe group consisting of C₁ to C₃ alkyl, halo-substituted C₁ to C₃ alkyl,methoxy-substituted C₁ to C₃ alkyl, and hydroxyl-substituted C₁ to C₃alkyl; wherein the sieving component and the surface interactioncomponent are the same or different; and wherein the composition doesnot include a crosslinked polymer gel.
 9. The composition of claim 1wherein the sieving component is linear polyacrylamide.